APOBEC3G, in the absence of the HIV-1 accessory Vif gene, is packaged into HIV-1 particles during assembly, apparently through the formation of a complex with the RNA-recruiting nucleocapsid region of the Gag viral protein [1,2]. APOBEC3G subsequently associates with the viral reverse transcription complex, where it deaminates cytidine residues to uridine in the nascent minus-strand viral DNA. These deoxyuridine-rich transcripts are then either degraded or serve as templates for the synthesis of plus-strand DNA, thus yielding proviruses that are largely non-functional as a result of G → A hypermutation [3–5]. The HIV-1 Vif protein counteracts the antiretroviral activity of APOBEC3G by inducing its ubiquitination and degradation .
As APOBEC3G is an important host factor that may confer an intrinsic block to HIV-1, the APOBEC3G gene was screened in American  and European  cohorts to identify genetic variants that might influence the progression of AIDS. Twenty-nine single nucleotide polymorphisms (SNP) with frequencies of 1% or greater were identified in the APOBEC3G gene. Of these, three mutations are located in the 5′ putative regulatory region and three SNP are located in exons. The T → C variant at codon 119 (exon 3) leads to a synonymous change and does not modify the amino acid composition of the protein. The A → G at codon 186 (exon 4) and the C → G at codon 275 (exon 6) substitutions result in amino acid changes from histidine to arginine (H186R) and glutamine to glutamic acid (Q275E), respectively. The H186R variant was strongly associated with a decline in CD4 T cells and accelerated progression to AIDS-defining conditions in African Americans, but not in American and European Caucasians [7,8].
As APOBEC3G could possibly confer resistance to HIV-1 infection, we investigated the effects of both APOBEC3G regulatory and coding region variants that may modify the protein's expression or function in a cohort of highly exposed Caucasians to HIV-1 for their influence on the susceptibility to infection.
The study population consisted of 122 Caucasian individuals exposed to HIV enrolled in prospective cohort studies in Montreal. We analysed the DNA samples of 69 individuals infected with HIV-1 by homosexual contact or injection drug use recruited during primary infection. We identified 53 exposed seronegative (ESN) individuals matched for age and sex, from whom DNA samples were available from prospective studies on the basis of their continued seronegative status for at least 5 years, despite high-risk sexual behaviours or documented exposure to contaminated blood products . The DNA samples of these ESN individuals were analysed as the control group (HIV negative). APOBEC3G genotyping was carried out by DNA direct sequencing procedures. The polymerase chain reaction primers were designed to cover the entire putative promoter region and exons 3, 4 and 6, as well as their exon–intron boundaries that are important for messenger RNA splicing. Primer sequences and conditions are available upon request. Control subjects were also genotyped for the CCR5 gene, and none were found to be homozygous for the 32-base pair deletion allele. The study was approved by ethics committees, and all participants gave written informed consent.
Table 1 shows the allelic distribution of the APOBEC3G polymorphisms among HIV-infected and ESN subjects. The overall APOBEC3G allelic distribution was similar to that observed in other populations, with two notable differences. The F119F variant was observed more frequently in our sample population (37%) than in African Americans (8%), whereas the H186R was less prevalent (≅3%) in our cohort than in African Americans (37%) [7,8]. There was no difference in allelic frequencies for each APOBEC3G promoter and coding region SNP among HIV-infected and ESN subjects. However, a novel C40693T variant in intron 4 of APOBEC3G was significantly associated with an increased risk of HIV-1 infection (P = 0.03).
The C40693T nucleotide variant is located 68 bp downstream of the exon 4 natural 3′ splice site and could potentially affect mRNA splicing by creating an alternative acceptor splice site. This situation could lead to the production of aberrant mRNA and the absence of functional APOBEC3G protein. There are over a hundred different examples of point mutations that lie in the vicinity of mRNA splice junctions and that have been held responsible for human disease by altering the efficacy of mRNA splicing . Nevertheless, further studies are needed to investigate whether this mutation could indeed cause aberrant APOBEC3G splicing at the mRNA level.
We cannot rule out the possibility that the observed association between APOBEC3G intron 4 polymorphism and HIV infection might be caused by strong linkage disequilibrium to other sequence variations in the APOBEC3G gene or other linked genes. The APOBEC3 gene complex is a cluster of eight genes (APOBEC3A to 3H) arrayed in tandem on human chromosome 22 . Additional members of the human APOBEC family are endowed with activity against HIV-1. For example, APOBEC3B and APOBEC3F, which is largely co-expressed with APOBEC3G, exhibit moderate levels of activity against HIV-1 [12,13], and might both play a role in susceptibility to HIV infection. Moreover, extensive screening of HIV exposed-uninfected and infected cohorts revealed that homozygosity for the 32-bp deletion at the CCR5 locus conferred significant protection against infection in homosexual individuals. This was not the case in our study because none of the high-risk exposed uninfected individuals were homozygotes for this allele.
Our study focused on a well-characterized group of individuals that were carefully assessed for exposure, and demonstrated that a novel genetic variant in intron 4 of APOBEC3G is significantly associated with an increased risk of acquiring HIV-1 through homosexual contacts or injection drug use. Further studies are needed to define the net impact of this mutation on the susceptibility to HIV-1 infection.
Sponsorship: This work was supported by the Réseau Sida et Maladies Infectieuses du Fonds de la Recherche en Santé du Québec (FRSQ). M.R. and M.A. are supported by career awards from FRSQ.
1. Alce TM, Popik W. APOBEC3G is incorporated into virus-like particles by a direct interaction with HIV-1 Gag nucleocapsid protein. J Biol Chem 2004; 279:34083–34086.
2. Cen S, Guo F, Niu M, Saadatmand J, Deflassieux J, Kleiman L. The interaction between HIV-1 Gag and APOBEC3G. J Biol Chem 2004; 279:33177–33184.
3. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt I, Watt IN, et al
. DNA deamination mediates innate immunity to retroviral infection. Cell 2003; 113:803–809.
4. Lecossier DF, Bouchonnet F, Clavel F, Hance AJ. Hypermutation of HIV-1 DNA in the absence of Vif protein. Science 2003; 300:1112.
5. Mangeat B, Turelli P, Caron G, Freidli M, Perrin L, Trono D. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 2003; 424:99–103.
6. Sheehy AM, Gaddis NC, Malim MH. The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med 2003; 9:1404–1407.
7. An P, Bleiber G, Duggal P, Nelson G, May M, Mangeat B, et al
. APOBEC3G genetic variants and their influence on progression to AIDS. J Virol 2004; 78:11070–11076.
8. Do H, Vasilescu A, Diop G, Hirtzig T, Heath SC, Coulonges C, et al
. Exhaustive genotyping of the CEM15 (APOBEC3G) gene and absence of association with AIDS progression in a French cohort. J Infect Dis 2005; 191:159–163.
9. Makedonas G, Bruneau J, Alary M, Tsoukas CM, Lowndes CM, Lamothe F, et al
. Comparison of HIV-specific CD8 T-cell responses among uninfected individuals exposed to HIV parenterally and mucosally. AIDS 2005; 19:251–259.
10. Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90:41–54.
11. Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, Scott J, et al
. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 2002; 79:285–296.
12. Liddament MT, Brown WL, Schumacher AJ, Harris RS. APOBEC3F properties and hypermutation preferences indicated activity against HIV-1 in vivo
. Curr Biol 2004; 14:1385–1391.
13. Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, Malim MH. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol 2004; 14:1932–1936.